Regulating gene expression either by increasing expression or decreasing expression is considered beneficial for treatment of human diseases. This is especially important in those diseases in which master regulatory genes have been identified. While a majority of efforts have been extended toward enhancing gene expression, down-regulating specific gene expression is equally important. A naturally occurring gene-silencing mechanism triggered by double-stranded RNA (dsRNA), designated as small interfering RNA (siRNA), has emerged as a very important tool to suppress or knock down gene expression in many systems. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21-25 nucleotide fragments with characteristic 5′ and 3′ termini (Provost, P. D. et al. Embo J, 2002, 21:5864). These siRNAs act as guides for a multi-protein complex, including a PAZ/PIWI domain containing the protein Argonaute2, that cleaves the target mRNA (Hammond, S. M. et al. Science, 2001, 293:1146-1150). These gene-silencing mechanisms are highly specific and potent and can potentially induce inhibition of gene expression throughout an organism.
The last decade has seen tremendous progress in gene expression technology, including the continued development of both non-viral and viral vectors. The non-viral approach to gene expression involves the use of plasmid DNAs (pDNAs) which have a number of advantages, including ease of use and preparation, stability and heat resistance, and unlimited size. The plasmids do not replicate in mammalian hosts and do not integrate into host genomes, yet they can persist in host cells and express the cloned gene for a period of weeks to months.
Furthermore, several investigators have utilized a replication-deficient episomal adenovirus as a vehicle for transient gene expression. Adenoviral vectors are very efficient at transducing target cells in vitro and in vivo and permit transgene expression in a dose-dependent manner (Stampfli, M. R. et al., J Clin Invest, 1998, 102:1704-1714; Walter, D. M. et al., J Immunol, 2001, 166:6392-6398). However, adenoviral vectors can cause acute inflammation and an immune response to viral vector-encoded antigens. While efforts to improve viral vectors continue, acute inflammation and immunogenicity to viruses remain the major stumbling blocks to the application of viral-mediated gene transfer. The adeno-associated virus has been considered particularly advantageous because it is non-pathogenic in humans and it causes less inflammation and immunogenicity than adenovirus.
In order to utilize siRNA technology in mammalian cells, gene transfer methods must be employed. However, a majority of common chronic diseases and viral infections are multifactoral and it is difficult to effectively treat a disease by only silencing one particular gene. It would not only be advantageous to create vectors that employ siRNA technology to target one specific gene responsible for a disease, but to create a single vector capable of targeting and down-regulating several genes responsible for multifactoral diseases. Alternatively, in some multifactoral diseases, it would be advantageous to simultaneously express certain endogenous genes, such as endogenous tumor suppressor genes, or exogenous genes, such as exogenous apoptotic genes.
Infection by Dengue Virus (DV, also referred to herein as DEN) is one type of viral infection where siRNA technology would be advantageous. The need for safe and effective inhibition of DV infection (e.g., prophylaxis or treatment), a category A mosquito-borne human pathogen, is a critical global priority. DV causes dengue hemorrhagic fever/dengue shock syndrome (DBF/DSS), which is associated with heterologous secondary DV infection and affects thousands of people worldwide. Moreover, the incidence of DHF/DSS is increasing in the Western Hemisphere. Currently, there are no specific antiviral treatments available. Although many different approaches are being taken to develop prophylactic DEN vaccines, none have been licensed for public health use.
DV belongs to the family of Flaviviruses and is an enveloped single plus-stranded RNA virus with four distinct serotypes. The DEN genome of approximately 11,000 nucleotides encodes a polyprotein (C-prM-E-NS1-NS2a-NS3-NS4a-NS4b-NS5) consisting of three structural proteins (C, prM and E) and seven nonstructural proteins. The open reading frame is flanked by a 100 nucleotide-long noncoding region (NCR) at the 5′ end and a 400 to 600 nucleotide-long NCR at the 3′end (Lindenbach, B. & Rich, C. M. Fields Virology, Lippincott Williams & Wilkins. 2000). Although the mechanism of DV pathogenesis is not completely clear, DV typically appears to replicate locally in skin or blood dendritic cells (DCs) and secondary infection and by antibody-dependent enhancement (ADE) may also involve DCs, monocytes, and macrophages. An in vivo gene-silencing approach using siRNA to decrease DV replication is an effective antiviral approach aimed at attenuating the DV viral burden and potentially protecting infected subjects from DHF/DSS.
The mechanism of siRNA is a well-characterized phenomenon that has proven effective in silencing a number of genes of different viruses including those of DV (Fire, A. Trends Genet, 1999, 15:358; Blair, C. D. et al. Clin Microbiol Rev, 2000, 13:651; Adelman, Z. N. et al. Insect Mol Biol, 2001, 10:265; Caplen, N. J. et al. Mol Ther, 2002, 6:243). The plus-sense RNA genome of DV must first be translated into the viral polyprotein for infection to occur, and this makes it an excellent target for siRNA. Plasmids encoding antisense DV RNA could be introduced into cells prior to infection, and when dengue virions enter and their RNA is uncoated, the complementary interfering RNAs bind to it and cause cleavage by a host RNase. Adelman et al. (Adelman, Z. N. et al. Insect Mol. Biol., 2001, 10:265-273) transfected mosquito cells with a plasmid encoding a DV-2 inverted RNA repeat, and subsequently challenged them with DV-2. Production of viral envelope protein and genomic RNA was greatly reduced in those cells expressing the RNA construct. Small (˜20 nt) RNAs were identified in the cells consistent with the activation of the RNA-interference system. Although the method has only been used to block DV replication in insect cells, siRNA is a workable strategy for in vivo protection from DV and other viral infections. siRNA oligonucleotides directed against the target region near the translation initiation site of DV virus RNA were the most effective at blocking replication (Caplen, N. J. et al. Mol Ther, 2002, 6:243).